Accumulating evidence suggests that oxidant stress contributes to the pathogenesis of hypertension and atherosclerosis. Vascular NAD(P)H oxidases are activated in hypertension, predominantly contribute to vascular production of reactive oxygen species (ROS). Indeed, scavenging of ROS with either antioxidant enzymes or inhibitors of vascular NAD(P)H oxidases significantly restores endothelial function to reduce blood pressure. Nevertheless, these treatments failed to completely normalize endothelial function or blood pressure, implicating involvement of other mechanisms that are not readily reversible by scavenging ROS. Recent studies demonstrated that in atherosclerotic or hypertensive blood vessels, endothelial nitric oxide synthase (eNOS) transformed from an antioxidant enzyme into a pro-oxidant enzyme producing ROS rather than nitric oxide. 1 of the causes for this uncoupling of eNOS seems to be a deficiency in eNOS cofactor tetrahydrobiopterin (H4B). It is interesting to speculate that unsatisfactory outcomes of some antioxidant therapies are partially due to their ineffectiveness in recoupling eNOS. The precise mechanisms as to how H4B becomes persistently deficient under disease conditions remain unclear. We and others have recently shown that angiotensin II uncouples eNOS in vitro and in vivo (Figures 1-3, Mollnau et al Cir Res 90: E58-65). In this project we will fully characterize this phenomenon and determine whether it is mediated by vascular NAD(P)H oxidase-derived hydrogen peroxide and an endothelial H4B deficiency (aim 1). In aim 2 we will determine whether angiotensin II modulation of endothelial H4B salvage enzymes contributes to endothelial H4B deficiency. Effects on eNOS recoupling, endothelial function and blood pressure of H4B precursors/analogues, inhibition of vascular NAD(P)H oxidases, overexpression of H4B salvage enzymes or combination of 2 or more will be studied in angiotensin II infused mice. In aim 3 we will examine potential roles of eNOS monomerization, threonine phosphorylation and dissociation from heat shock protein 90 (HSP90) in angiotensin II uncoupling of eNOS and their interdependence with H4B deficiency. Effects on eNOS recoupling of agents promoting eNOS dimerization and its association with HSP90 will be studied. These proposed experiments could ultimately lead to novel therapeutics restoring nitric oxide production from uncoupled eNOS, which are anticipated to reduce blood pressure and impede atherosclerosis. We will also determine whether eNOS uncoupling augments hypertension (aim 4, H4B-deficient hph-1 mice will be studied). This will address the question: is eNOS uncoupling a mere consequence/marker of hypertension or 1 of the causes?